Extended Data Fig. 1: Screening of unstable NbALFA.
a, Sequence alignment of the amino acids of NbALFA with other Nbs susceptible to degradation, including αCA dNb6mut, αDHFR dNb6mut, and dGBP1. Amino acid positions are numbered according to the ImMunoGeneTics information system (IMGT), FR: framework, CDR: complementarity determining region. b, Normalized fluorescence intensities of NbALFA-EGFP and 6mutNbALFA-EGFP, with or without the ALFA tag, were measured in U2OS cells transiently transfected with either NbALFA-EGFP or 6mutNbALFA-EGFP, along with mCherry-FLAG or mCherry-FLAG-ALFA plasmids. Quantitative analysis from three biologically independent experiments using Fiji software normalized fluorescence intensities to that of NbALFA-EGFP with mCherry-FLAG transfection. Data are expressed as the mean ± s.e.m. Significance was calculated by two-tailed Student’s t-test; *p < 0.05 (p = 0.0120),***p < 0.0001 (p = 0.0009). c, Validation of the ubiquitin fusion technique in 293T cells transiently transfected with either Ub(G76V)-(M)NbALFA-mEmerald or Ub-(M)NbALFA-mEmerald. Whole-cell lysates were prepared, and protein samples were separated by SDS-PAGE, transferred to PVDF membranes, and probed with anti-EGFP and anti-β-actin antibodies. Protein expression was visualized with enhanced luminescence reagents.
